Analysis of BlaEC family class C beta-lactamase

Abstract Recent years have witnessed an increased prevalence of intrinsic and acquired beta-lactamase-producing bacteria, severely limiting human and veterinary medicine therapeutic options. The present study aimed to design specific oligonucleotides for rapid PCR detection of the cephalosporinase-encoding gene blaEC (BlaEC family class C beta-lactamase). A total of three primers were designed to detect 2281 variants of the blaEC gene and two sets of primer pairs were also tested against DNA from 11 strains. The study indicates that the proposed primers should be able to detect 100% of all described blaEC genes in different bacterial strains and monitor their spread. After comparing the amino acid sequences, a phylogenetic tree was created based on the presence of conserved amino acids and homologous motifs. More than 24 760 mutations in BlaEC enzymes have been identified. The mutations involving 371 amino acid positions and these hotspots can change the structure and activity of the monitored enzymes. We predicted several BlaEC enzymes with a broadened substrate activity against higher-generation cephalosporins.


Introduction
An influential group of enzymes that allow bacteria to resist the effects of beta-lactam antibiotics are beta-lactamases ( bla ) of the AmpC type .T hese are cephalosporinases that hydr ol yze most penicillins , cephalosporins , oxyimino-cephalosporins , and monobactams .T hese antibiotic groups contain frequently applied antibiotics, and the resistance of bacterial pathogens to their effect r epr esents a serious clinical pr oblem.In the case of continuous production of the mentioned enzymes, the minimum inhibitory concentration (MIC) values are increased above the clinical breakpoints of the respective antibiotics.Another problem is the possibility of switc hing fr om inducible to continuous production of the enzymes during antibiotic administr ation, whic h can lead to treatment failure and, in the case of sepsis, to the death of the patient.Classic beta-lactamase inhibitors (clavulanic acid and sulbactam) have zero effect in the case of this group.
AmpC beta-lactamases encoded by c hr omosomal genes ar e widely distributed in most bacterial members of the order Enterobacterales as well as bacteria from other families, such as Acinetobacter spp., Aeromonas spp., or Pseudomonas spp.In many bacteria, c hr omosomal ampC gene expression is usually low.The expression is induced at the time of response to an external stimulus, such as a beta-lactam antibiotic.Individual beta-lactam antibiotics differ in their ability to induce AmpC expression, with strong inducers including penicillins and first-generation cephalosporins (J acob y 2009 ).Beta-lactamase inhibitors also act as inducers, especially clavulanic acid (Weber and Sanders 1990 ). Further, AmpR, AmpD, and AmpG (cell wall metabolism pr oteins) ar e involv ed in the induction of the ampC gene (Hanson and Sanders 1999 ).The cytosolic protein AmpD, which acts as a negative expression regulator, is also involved in regulating AmpC expression (Jacobs et al. 1995 ).
According to the Ambler classification scheme, these betalactamases are classified in class C and, according to the Bush-J acob y-Medeiros classification, they belong to group 1 (Bush 2013, Bush and J acob y 2010, Harris and Ferguson 2012 ).The new serine enzymes of subgroup 1e are variants of group 1 with greater activity against ceftazidime and other oxyimino-lactams due to amino acid substitutions, insertions or deletions.They have been designated as extended-spectrum AmpC (ESAC).
The main objectives of our study were to design specific oligonucleotides for r a pid PCR detection and to monitor the distribution of all pr e viousl y described BlaEC enzymes among bacteria.Last but not least, we aim to provide more comprehensive information about this group of enzymes.

Primer design and in silico analysis of BlaEC sequences
A total of 2281 sequences of genes encoding BlaEC enzymes, containing only the coding regions without their promoters, described in the BLDB database (last accessed on 2 May 2023) (Naas et al. 2017 ) were downloaded from the GenBank database ( http: // www.ncbi.nlm.nih.gov/genbank ).The bioinformatics software Geneious Prime 2023.2.1 (Biomatters, New Zealand) (Kearse et al. 2012 ) was used to compare nucleotide and amino acid sequences.The blaEC nucleotide sequences and their amino acid sequences were aligned using MUSCLE algorithm (Edgar 2004 ) and Geneious alignment (default settings in both) as implemented in Geneious Prime, r espectiv el y.Pr otein statistics generated in Geneious within the sequence alignments at the Cost Matrix of Blosum62 were used to study amino acid substitutions.
Primers were designed as described pr e viousl y (Ml ynarcik et al. 2021b ).

Mutation frequency studies
The mutation frequency for a given site is defined as the number of sequences that have a mutation (a different amino acid listed in the BlaEC-1 r efer ence sequence) on that site .T he numbering of the amino acid residues is by the structural alignment-based numbering of class C beta-lactamases or the SANC scheme (Mack et al. 2020 ).

Testing specificity of designed primers with in silico primer binding tests and conventional PCR
In silico binding tests were performed to e v aluate the specificity of three primer pairs.All primers were tested against blaEC nucleotide sequences using the Test with Saved Primers option in Geneious .T he specificity of BlaEC-F1/R1 and BlaEC-F2/R2 primers was also tested with conventional PCR using genomic DNA extr acted fr om E. coli isolates.For the pr epar ation of the r eaction mixture (25 μl), we used Combi PPP Master Mix (Top-Bio Pr a gue, Czech Republic) as described by the manufacturer.The PCR samples were examined using a 1% a gar ose gel.The gel was treated with SYBR Safe dye from Invitrogen and observed under a UV transilluminator for visualization.

Phylogenetic tree construction
A maxim um-likelihood phylogenetic tr ee was gener ated and visualized using the protein alignment with PhyML (Guindon et al. 2010 ) implemented within Geneious Prime with the Le-Gascuel substitution model and without bootstr a pping.

Results
Our study attempted to design primers to detect 2281 variants of blaEC genes found in Esc heric hia and Shigella strains based on the BLDB database.Inter estingl y, a further BLASTn search of the blaEC genes against the NCBI database identified a series of enterobacteria carrying the same beta-lactamase type (e.g.class C betalactamase BlaEC-5 in Enterobacter hormaechei -CP056649, BlaEC-1 in Salmonella spp .-CP046033), including the described blaEC-1and blaEC-243 -harboring plasmid in Klebsiella oxytoca (CP069925) and E. coli (JN412137), r espectiv el y.Inter estingl y, in the latter plasmid, we found that two mobile elements, including the insertion sequence IS 10 (upstream) and IS CR2 (do wnstream), w ere located in the vicinity of the blaEC-243 (Fig. 1 -I).In addition, in another E. coli strain (AY559027), IS 10 flanked by the 9-bp direct repeat sequences (CGTTTTGTA) were inserted between the ampC attenuator region and the start codon of the partial blaEC-like sequence (Fig. 1

-II).
A total of three specific primer pairs were created (Table 1 ) using Primer3 (Geneious).The primer-BLAST results sho w ed that these oligonucleotides could detect all allelic variants of this betalactamase type.Briefly, the BlaEC-F1/R1 primers described earlier (Mlynarcik et al. 2021a ) could capture 206 specific blaEC variants.Another 139 specific variants could be tested using the BlaEC-F2/R2 primers.In addition, both primer pairs can jointly detect another 1927 BlaEC v ariants, corr esponding to amplified PCR products, using DNA from tested isolates, as described below.The remaining nine distinct subtypes of BlaEC enzymes could be verified using the specific BlaEC-F3/R3 primers .T he specificity of the first two primer pairs was also analyzed by means of PCR analysis.PCR performed on DNA from Escherichia isolates using BlaEC-F1/R1 and BlaEC-F2/R2 primer sets produced DNA fragments of the expected sizes (307 and 761 bp; Figure S1, Supporting Information ) and confirmed the target specificity of the primer pairs.
In addition, a point mutation study sho w ed that 24 766 amino acid c hanges wer e detected in BlaEC enzymes .T he most common amino acid changes recorded were as follows: A → T (1807 times), D → A (1543 times), T → A (1478 times), E → D (1477 times), and K → N (1388 times).By contrast, amino acid c hanges r ecorded onl y once were found in 21 cases, such as A → C or F or L, D → C or P or S, E → N or Y, F → K, G → Q, and I → R.More detailed information about point mutations in BlaEC enzymes is given in Table S2 (Supporting Information) .
Figure 2 shows the mutational frequencies for each position, highlighting evolutionary hotspots.It shows many hotspots along multiple sequence alignments covering more than 24 760 mutations, of which 27 hotspots had more than 200 mutations .T he frequency of mutations was high at residue positions Asp351, Lys239, Leu241, Thr89, Leu238, Glu245, Ser282, Gln235, Arg232, and Ala220 (2159,1368,1350,1238,1188,1184,1108,1101,955, and 854 mutations, r espectiv el y) (data not shown).These data suggest that beta-str ands m utated less fr equentl y and a ppear to be mor e r esistant to mutations than helices.Table 2 illustrates predicted extended-spectrum BlaEC enzymes based on specific amino acid changes involved in the broadening of the hydrolysis spectrum described by Doi et al. ( 2004 ) and Mammeri et al. ( 2006 ).Based on their findings, we considered a total of four amino acid changes within the entire BlaEC enzyme sequences at positions 287, 296, 298, and 350 (S-287- * , H/R-296- * , V-298- * , and V-350- * ) and one deletion of three amino acids (GSD) at positions 286-288 (286-GSD-288); thus, we identified 16 ESAC enzymes.By considering other amino acid changes at these positions, we get a total of 58 ESAC enzymes.Regarding the PCR detection of these 58 ESAC genes, our second primer, desig-nated as BlaEC-F2/R2, can ca ptur e all the mentioned variants except blaEC-861 , which, on the other hand, detects the BlaEC-F1/R1 primer.
Using a phylogenetic tr ee, r elationships between BlaEC enzymes were illustrated based on the similarity of their amino acid sequences.A rooted phylogenetic tree allo w ed us to identify several major groups and subgroups (Fig. 3 ).

Discussion
According to data from the BLDB database, oxacillinases and the BlaEC most abundant class C beta-lactamases r epr esent mor e than 48% (3419 variants) of all beta-lactamases described (Naas et al. 2017 ).Ther efor e, we focused on this particular type of ampC genes.At the same time, Doi et al. ( 2004 ) and Mammeri et al. ( 2006 ), in their r espectiv e studies, detailed se v en BlaEC enzyme variants of clinical relevance that show an extended capacity to act on higher-generation cephalosporins.More detailed information about PCR detection of oxacillinases in bacteria is available else wher e (Ml ynarcik et al. 2020 ).
The cause of bacterial resistance is a mutation in one of the structur al or r egulatory genes involv ed in the expr ession of betalactamases of the AmpC type and the subsequent switch from inducible to permanent production.In addition, overproduction can also result from insertions in the AmpC promoter and replacement of the native promoter region with a promoter from other bacteria, ultimately allowing for higher levels of gene expression (P a panicolaou et al. 1990 ). Furthermore, the horizontal acquisition of AmpC beta-lactamases has r ecentl y gained importance and r epr esents an essential driving force in incr easing r esistance (Mac Aogain et al. 2016 ).Considering AmpC beta-lactamases of the BlaEC, a large number of these variants have been described, but only some of them ar e expr essed to a sufficient extent to cause antibiotic resistance .For example , as shown in Fig. 1 , genes encoding BlaEC production can be expressed using functional promoters r epr esented by insertion sequences, as for oxacillinases (Mlynarcik et al. 2020 ).
Inter estingl y, hyper pr oduction of the AmpC c hr omosomal enzyme, combined with the loss of outer membrane protein, has been found to play a role in de v eloping carba penem r esistance in Serratia marcescens (Suh et al. 2010 ).An increasing number of papers have described the synergy between AmpC beta-lactamase pr oduction, efflux pump ov er expr ession and low outer membrane permeability in clinical isolates.For example, Tomas et al. found that Pseudomonas aeruginosa isolates from cystic fibrosis patients that ov er expr essed mexA or ampC and reduced oprD were associated with beta-lactam resistance (Tomas et al. 2010 ).Additionally, Liu and colleagues found that low expression of outer membrane porin with cephalosporinase ov er expr ession or extended-spectrum beta-lactamase (ESBL) production and efflux pump ov er expr ession may contribute to carbapenem resistance in carba penem-insensitiv e Enterobacter cloacae isolates that produce noncarbapenemase in the hospital (Liu et al. 2021 ).Therefore , detecting all AmpC beta-lactamases , including BlaEC enzymes, is necessary to better understand the de v elopment of multidrug resistance.
Recentl y, we hav e designed se v er al primers to monitor ESBL genes in clinically significant bacteria (Mlynarcik et al. 2021a ,b ), focusing on another group of extended-spectrum BlaEC enzymes in this study.Three primers (Table 1 ) were designed to detect 2281 variants of blaEC genes in enterobacteria.Ho w ever, the first two pairs of primers can detect 2272 variants of the monitored genes, r epr esenting mor e than 99.6% of the total number of subtypes described.Since the plasmid-encoded blaEC genes have already been described in enterobacteria, the primers we designed can be used to monitor these resistance genes.
The dataset contained more than 24 760 mutations covering a total of 371 out of 378 residue positions of BlaEC enzymes, her e r eferr ed to as m utational hotspots .T hese hotspots had at least one mutation.In particular, the data sho w ed that hotspots located in helices were most fr equentl y m utated (Fig. 2 ).In addition, proline substitutions are also shown in this figure, as it has been reported in other studies that proline represents the most disruptive substitution (disrupts enzyme function), and methionine is most tolerated (Gray et al. 2017, Vakulenko et al. 2002 ).
Further, the m utational anal ysis study suggests that at least 16 subtypes of BlaEC enzymes r epr esent ESAC beta-lactamases taking into account the conclusions made by Doi et al. ( 2004 ) and Mammeri et al. ( 2006 ).Regarding the PCR detection of these ESAC genes, all these variants could be detected using our first primer, BlaEC-F1/R1.When considering other amino acid changes (Table 2 ), such as the change from serine to isoleucine or arginine at position 287, the estimated number of ESAC enzymes is 58, but experimental data are needed to support this speculation.The PCR primer F2/R2 reported in this study would allow the detection of 98.3% of ESAC variants (57/58) and would not be able to detect BlaEC-861.This subtype could be verified using a specific BlaEC-F1/R1 primer.In addition, there may be many more of these enzymes .T her efor e, we tried to summarize the frequency of all amino acid changes along the entire sequence in all 2281 variants of BlaEC enzymes .T he abo ve results could be helpful for scientific personnel working in the field of these beta-lactamases and their drug resistance analysis.
This study highlights the diversity of BlaEC beta-lactamases, and our data may help define the boundaries of the BlaEC enzyme subfamily.On the other hand, these least similar BlaEC variants ( Table S3, Supporting Information ) may also r epr esent a ne w type of beta-lactamases.As was the case, e.g. with KLUC-1 and the CTX-M-1 subgroup of beta-lactamases, whic h shar ed 85%-86% amino acid identity (Decousser et al. 2001 ).And the peculiar hydr ol ysis pr ofile may also be r elated to this.
PCR and whole genome sequencing (WGS) are valuable tools for screening and monitoring clinically important bacteria producing ESBL and carbapenemases.Ho w ever, each of these methods has adv anta ges and limitations, and the choice between them depends on the specific objectives and resources of the laboratory or r esearc h pr oject.
PCR is particularl y cost-effectiv e and helpful in labor atories without access to next-generation sequencing platforms or extensiv e financial r esour ces.It offers lo w er cost, faster turnaround time and the ability to detect specific r esistance genes, suc h as blaEC genes detected in v arious enter obacteria and on plasmids.This is critical for understanding the evolution of multidrug resistance in bacteria due to the coexistence and cotransmission of beta-lactamases with other resistance genes.Ho w ever, PCR is mor e tar geted and, unlike WGS, may not detect other r esistance genes or genetic variations not explicitl y tar geted by the primers used in the PCR assay.On the other hand, WGS r equir es mor e r esources and ca pacity but offers a compr ehensiv e anal ysis of genetic relationships.It enables the detection of all potential resistance genes.It provides a more complete picture of the bacterial genome .It ma y help better understand the epigenetic mechanisms of phenotypic resistance, enabling clinicians to make r a pid clinical decisions that impr ov e patient health outcomes.In addition, it may contribute to the discovery of novel resistance mechanisms.Ho w ever, WGS can be more expensive, requires more sophisticated equipment and bioinformatics expertise, and takes longer to obtain results.For example, the actual inter pr etation of the results and the large computer memory needed to pr ocess and stor e the genomic data r emain significant c hallenges.
In summary, our results suggest that the tested primers could be used for PCR to detect and monitor the spread of these genes into other bacterial species .T he study also analyzed mutational fr equencies, identified e volutionary hotspots and conserv ed amino acids within the BlaEC enzymes studied, and predicted extended-spectrum BlaEC enzymes based on specific amino acid c hanges.A phylogenetic tr ee was used to illustrate relationships between BlaEC enzymes based on their amino acid sequence similarity.

Ac kno wledgments
This r esearc h was funded by IGA_LF_2023_012 project and the project National Institute of Virology and Bacteriology (Progr amme EXCELES, ID Pr oject number LX22NPO5103)-Funded by the European Union-Next Generation EU.

Figure 2 .
Figure 2. Amino acid changes along the primary sequence of BlaEC enzymes, shown with evolutionary conservation.The observed amino acid substitutions in 2280 BlaEC enzyme variants are above the BlaEC-1 enzyme sequence.Proline substitutions are highlighted in red.The colored bars c har acterize the general categories of amino acids.Residues without bars represent zero identified substitutions.Numbers are read vertically and indicate SANC-based amino acid residue positions .Abo ve the observed amino acid substitutions are the change frequencies for each position.Alpha-helices (blue barrel), 3 10-helix (green barrel), and beta-strand or beta-bridge (y ello w arro ws).

Figure 3 .
Figure 3.A phylogenetic tree obtained by comparing 2281 BlaEC enzymes using Geneious PhyML.Characteristic BlaEC enzymes are indicated at the end of the br anc hes.Red circles r epr esent some sequence types with the lowest amino acid identity.The 58 putative extended-spectrum BlaEC enzymes are highlighted in blue.

Table 1 .
Primer sequences used to detect the bla EC genes by PCR.